The invention relates to probes constituted by polynucleotides, more precisely oligonucleotide fragments, comprising, as purine bases, adenine groups modified so that they are capable of forming three hydrogen linkages with reactive groups of thymine, a process for their production, as well as their uses.
It is now well known to specialists to use for certain analyses techniques based on hybridization between a predetermined sequence of nucleotides used as a probe and a nucleotide sequence complementary to those of the probe. These techniques are especially useful for the detection of particular nucleotide sequences, such as DNA or RNA sequences and the isolation of such sequences. The nucleic acid sequence may possibly be contained in a composition containing nucleic acids. The practice of these techniques, in particular the obtaining of probes that are sensitive and capable of hybridizing stably with the complementary sequences, still poses problems.
Thus, one of the most frequent problems posed in genetic engineering is that of "fishing" for RNA messengers by synthetic oligodesoxynucleotides, that is to say the detection, isolation and analysis of messenger RNA sequences by hybridization with complementary DNA fragments (cDNA). This technique is based on the matching of A-T pairs (2 hydrogen bonds) and G-C pairs (3 hydrogen bonds).
The design and the synthesis of these probes are complicated by the degeneration of the genetic code: apart from the methionine (AUG) and the tryptophane (UGG) which necessitate a single codon, all the other amino acids are translated from 2 to 6 codons.
Until now, probes were obtained from a predetermined protein sequence by several strategies:
(a) Selecting in a sequence G in preference to A and T in preference to C. This gives to the hybridization either a stable pair, or a poor G-T match, which is called generally and in the following G-T "mismatch." This method has been applied with success to the synthesis of cDNA coding rat relaxin [P. Hudson et coll., Nature (1981) 291: 127], but is generalisable with difficulty by reason of its lack of sensitivity. PA1 (b) Preparing all the possible sequences with the corresponding ambiguities [B. E. Noyes et coll., Proc. Natl. Acad. Sci., USA, (1979), 76: 1770 and M. Mevarech et coll., J. Biol. Chem., (1979) 254: 7472], which can necessitate an enormous amount of work for the synthesis. PA1 (c) Synthesizing "mixed probes" with all the ambiguities on the same sequence. This technique has given good results for B-globin [R. B. Wallace et coll., Nucleic Acids Research (1981) 9: 879], for example, but represents for the organic chemist a "frustrating" side in view of the complexity of the mixture obtained and the number of trimers involved. This strategy does not always give satisfactory results because of the different reactivities of the various trimers involved at the level of each ambiguity. This leads to a lack of certainty as to the exact composition of the mixture obtained. PA1 can itself alone replace complex mixtures of sequences, and thus provide a considerable gain in time and money; PA1 has a well defined composition; PA1 has, with respect especially to the probes defined under (a) above, a better sensitivity in the detection after hybridization with complementary sequences and better stability of the hybrid thus formed (higher melting temperature); PA1 are capable of being used with identification methods based on immunological or even immuno-enzymatic reactions instead of radioactive detections, which are relatively insensitive and have well known drawbacks. PA1 when the adenine group must be modified on the C-2 position of the pyrimidine by a --NH.sub.2 group, 5'-O-dimethoxytrityl N,N'diisobutyryl 2-amino 2'-desoxy adenosine prepared from 2'-desoxy guanosine, by analogy with the technique described by W. L. Sung in J.C.S. (Comm), 1089 (1981) for thymidine; PA1 when the adenine group must be modified on the C-2 position of the pyrimidine by the --OH group, 5'-O-dimethoxytrityl, N-isobutyryl 2-hydroxy 2'-desoxy adenosine, which may be prepared also by analogy with the method of W. C. Sung, from the nucleoside derived from the corresponding xanthine; and PA1 when the adenine group must be modified on the C-2 position of the pyrimidine by an --SH group, 5'-O-dimethoxytrityl N-isobutyryl 2-mercapto 2'-desoxy adenosine, which may be prepared in the same manner as the corresponding 2-hydroxy derivative by replacing, by a known method, the --OH group by an --SH group. PA1 (1) a mixed probe comprising the three corresponding ambiguities (mixture of 8 hexadecamers): EQU 3' TAC TAC AT.sub.G.sup.A GT.sub.T.sup.C CT.sub.T.sup.C C 5'. PA1 (2) The specific sequence ##STR5## This sequence was prepared by means of dimers and trimers in the solid phase by the technique of H. Ito et coll, described in Nucleic Acids Research (1982), 10: 1755. In this sequence, when there was C/T degenerescence T was selected and when there was G/A degenerescence G was selected, for the reasons indicated above. In this selection process a "mismatch" was introduced on sixteen nucleotides (see below the actual structure of the cDNA). PA1 (3) The specific sequence ##STR6## according to the invention was prepared by means of monomers, dimers, and trimers in the solid phase by the above-indicated techniques with reference to this type of sequencing. In this sequence, the choice of degenerescence was the same as in sequece (2) and the adenosine A was replaced by 2-amino adenosine A*.